M. tuberculosis has infected 1.8 billion humans and accounts for 2 to 3 million deaths annually based on its ability to persist as an intracellular pathogen in human tissues. The host response to mycobacterial infection depends crucially on T cells, which until recently, were thought to be activated solely by peptide antigens bound to MHC class I and II proteins. The discovery of CD1 antigen presenting molecules shows how Langerhans cells and dendritic cells can activate T cells by presenting lipid antigens, including glucose monomycolate (Science 278, p. 283), mannosyl phosphomycoketides (Nature 404, p.884) and mycobactin- like lipopeptides (Science 303, p. 527). This proposal aims to use high performance liquid chromatography to isolate immunodominant antigens from the M. tuberculosis and M. leprae cell walls and then determine their structures using collision induced dissociation mass spectrometry, nuclear magnetic resonance and X- ray crystallography. Because mycobacteria remodel their cell walls and alter their antigen profiles during adaptation to intracellular growth, antigen discovery efforts will focus on pathogenic mycobacteria isolated directly from mammalian tissues and genetically modified M. tuberculosis that are deficient in the enzymes necessary for iron-scavenging from host tissues. The immunogenicity of each lipid antigen will be investigated by ex vivo analysis of T cell precursor frequencies in tuberculosis patients as measured by cytokine-capture ELISA, cell surface cytokine-capture immunofluorescence and staining with lipid-loaded CD1 tetramers. By measuring memory responses and the complexity of lipid antigen specificities in the CD1-restricted T cell repertoire, these studies will provide insight into the basic question of whether CD1 functions in the innate or acquired immune responses in humans. In addition, identification of the precise molecular structures of antigenic lipids offers the prospect of fundamentally novel, MHC-unrestricted immunodulatory drugs and vaccines against leprosy, tuberculosis and multi-drug resistant tuberculosis.